Activity of allelic variants of Pi class human glutathione S-transferase toward chlorambucil

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil, is often limited by the emergence of drug resistant tumor cells. Increased glutathione (GSH) conjugation (inactivation) of alkylating anticancer drugs or their activated metabolites due to overexpression of the Pi class GSH S-transferase (hGSTP1-1) is believed to be an important mechanism in tumor cell resistance to alkylating agents. Interestingly, the hGSTP1 locus is polymorphic in human populations and involves amino acid residues in positions 104 (isoleucine or valine) and/or 113 (alanine or valine). Here, we report that the allelic variants of hGSTP1-1 significantly differ in their efficiency in catalyzing the GSH conjugation of chlorambucil. Catalytic efficiency of the hGSTP1-1(I104,A113) isoform toward chlorambucil was approximately 2.5-, 7.5- and 15-fold higher compared with I104,V113, V104,A113 and V104,V113 variants of hGSTP1-1, respectively. The results of the present study suggest that hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to some alkylating agents.     

Purification and functional reconstitution of intact ral-binding Gtpase activating protein, RLIP76, in artificial liposomes.

We have recently shown that RLIP76, a ral-binding GTPase activating protein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi et al., Biochemistry 39,9327,2000). Transport function of RLIP76 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either that the residual intact RLIP76 was responsible for transport activity, or that the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLIP76 purified in buffers treated with either 100 or 500 microM serine protease inhibitor, PMSF. The purity and identity of recombinant and human erythrocyte RLIP76 was established by SDS/PAGE and Western-blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Higher PMSF concentration resulted in lower yield of the 38 kDa band and higher yield of intact RLIP76 from both human and recombinant source. In contrast, the substrate-stimulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent transport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaffected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Comparison of transport kinetics for daunorubicin between recombinant vs human erythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purified RLIP76 can modulate its transport activity.     

Tet system in the brain: Transgenic rats and lentiviral vectors approach

Local and regulated expression of exogenous genes in the central nervous system is one of the major challenges of modern neuroscience. We have approached this issue by applying the inducible tetracycline system to regulate the expression of EGFP reporter gene in double transgenic rats. We have obtained a strong induction of EGFP only in male testes, which correlated with a high level of rtTA expression only in this organ. To overcome the problem of lack of rtTA protein in the transgenic rat brain, we have delivered this Tet system activator with lentiviral vectors into the dentate gyrus of hippocampus of transgenic EGFP rats. As a result, after systemic application of doxycycline we have obtained inducible, stable and restricted to the desired brain region expression of EGFP. An advantage of this strategy is that the transgene is located in the same genetic milieu in every cell of the transgenic organism. This is crucial to obtain uniform expression of the regulated gene within the target brain structure. Combination of rat transgenesis and lentiviral vectors is a novel approach enabling precise spatiotemporal regulation of genes of interest strictly in the brain structure of choice or in other tissues. genesis 47:274–280, 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of the genotoxic potential of some microbial volatile organic compounds (MVOC) with the comet assay, the micronucleus assay and the HPRT gene mutation assay.

Microbial volatile organic compounds (MVOC), metabolites of fungi detected in indoor moulds and in working places in compost facilities are considered as a potential health hazard. Their toxicological relevance, however, is largely unknown and data are rare. The aim of this study was to evaluate in vitro the genotoxic, clastogenic and mutagenic potential of same typical MVOC. For the study of DNA damage human lung carcinoma epithelial A549 cells, V79 Chinese hamster fibroblasts and human peripheral blood cells were exposed and subjected to the alkaline comet assay (single cell gel test). Taking the Chinese hamster V79 cell line as a target clastogenic effects were studied by the micronucleus test and mutagenic effects by the hypoxanthine-guanine-phosphoribosyl transferase gene mutation test (HPRT test). The cytogenic effects of MVOC were assessed by a clonogenic assay using the A549 cell line. The alkylating agent methyl methanesulfonate (MMS) was taken as a positive control. The results indicate that MVOC induced DNA damage is only seen in conditions in which also cytotoxic effects are observed. Clastogenic and mutagenic effects could not be detected.     

Depletion of 4-hydroxynonenal in hGSTA4-transfected HLE B-3 cells results in profound changes in gene expression

Previously, we have shown that overexpression of 4-hydroxy-2-nonenal (HNE)-detoxifying enzyme glutathione S-transferase A4-4 (hGSTA4-4) in human lens epithelial cells (HLE B-3) leads to pro-carcinogenic phenotypic transformation of these cells [R. Sharma, et al. Eur. J. Biochem. 271 (2004) 1960-1701]. We now demonstrate that hGSTA4-transfection also causes a profound change in the expression of genes involved in cell adhesion, cell cycle control, proliferation, cell growth, and apoptosis, which is consistent with phenotypic changes of the transformed cells. The expression of p53, p21, p16, fibronectin 1, laminin gamma1, connexin 43, Fas, integrin alpha6, TGFalpha, and c-jun was down-regulated, while the expression of protein kinase C beta II (PKCbetaII), c-myc, cyclin-dependent kinase 2 (CDK2), and TGFbeta was up-regulated in transfected cells. These results demonstrate that HNE serves as a crucial signaling molecule and, by modulating the expression of genes, can influence cellular functions.     

The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase.

To further define the genes and gene products responsible for the in vivo conversion of phosphatidylglycerophosphate to phosphatidylglycerol in Escherichia coli, we disrupted two genes (pgpA and pgpB) which had previously been shown to encode gene products which carried out this reaction in vitro (T. Icho and C. R. H. Raetz, J. Bacteriol. 153:722-730, 1983). Strains with either gene or both genes disrupted had the same properties as the original mutants isolated with mutations in these genes, i.e., reduced in vitro phospholipid phosphatase activities, normal growth properties, and an increase in the level of phosphatidylglycerophosphate (1.6% versus less than 0.1% in wild-type strains). These results demonstrate that these genes are not required for either normal cell growth or the biosynthesis of phosphatidylglycerol in vivo. In addition, the total phosphatidylglycerophosphate phosphatase activity in the doubly disrupted mutant was reduced by only 50%, which indicates that there is at least one other gene that encodes such an activity and thus accounts for the lack of a dramatic effect on the biosynthesis of anionic phospholipids in these mutant strains. The phosphatidic acid and lysophosphatidic acid phosphatase activities of the pgpB gene product were also significantly reduced in gene-interrupted mutants, but the detection of residual phosphatase activities in these mutants indicated that additional genes encoding such phosphatases exist. The lack of a significant phenotype resulting from disruption of the pgpA and pgpB genes indicates that these genes may be required only for nonessential cell function and leaves the biosynthesis of phosphatidylglycerophosphate as the only step in E. coli phospholipid biosynthesis for which a gene locus has not been identified.     

Crystal structure of human glutathione S-transferase A3-3 and mechanistic implications for its high steroid isomerase activity

The crystal structure of human class alpha glutathione (GSH) S-transferase A3-3 (hGSTA3-3) in complex with GSH was determined at 2.4 A. Despite considerable amino acid sequence identity with other human class alpha GSTs (e.g., hGSTA1-1), hGSTA3-3 is unique due to its exceptionally high steroid double bond isomerase activity for the transformation of Delta(5)-androstene-3,17-dione (Delta(5)-AD) to Delta(4)-androstene-3,17-dione. A comparative analysis of the active centers of hGSTA1-1 and hGSTA3-3 reveals that residues in positions 12 and 208 may contribute to their disparate isomerase activity toward Delta(5)-AD. Substitution of these two residues of hGSTA3-3 with the corresponding residues in hGSTA1-1 followed by kinetic characterization of the wild-type and the mutant enzymes supported this prediction. On the basis of our model of the hGSTA3-3.GSH.Delta(5)-AD ternary complex and available biochemical data, we propose that the thiolate group of deprotonated GSH (GS(-)) serves as a base to initiate the reaction by accepting a proton from the steroid and the nonionized hydroxyl group of catalytic residue Y9 (HO-Y9) functions as part of a proton-conducting wire to transfer a proton back to the steroid. Residue R15 may function to stabilize the deprotonated thiolate group of GSH (GS(-)), and a GSH-bound water molecule may donate a hydrogen bond to the 3-keto group of Delta(5)-AD and thus help the thiolate of GS(-) to initiate the proton transfer and the subsequent stabilization of the reaction intermediate.     

A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A

Kinetochores are megadalton-sized protein complexes that mediate chromosome–microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1^CENP-U and Okp1^CENP-Q form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1–Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.     

Catalytic function of Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (GST-2) in conjugation of lipid peroxidation end products.

Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (earlier designated as GST-2) is related to sigma class GSTs and was previously described as an indirect flight muscle-associated protein with no known catalytic properties. We now report that DmGSTS1-1 isolated from Drosophila or expressed in Escherichia coli is essentially inactive toward the commonly used synthetic substrate 1-chloro-2,4-dinitrobenzene (CDNB), but has relatively high glutathione-conjugating activity for 4-hydroxynonenal (4-HNE), an electrophilic aldehyde derived from lipid peroxidation. 4-HNE is thought to have signaling functions and, at higher concentrations, has been shown to be cytotoxic and involved in the etiology of various degenerative diseases. Drosophila strains carrying P-element insertions in the GstS1 gene have a reduced capacity for glutathione conjugation of 4-HNE. In flies with both, one, or none of the GstS1 alleles disrupted by P-element insertion, there is a linear correlation between DmGSTS1-1 protein content and 4-HNE-conjugating activity. This correlation indicates that in adult Drosophila 70 +/- 6% of the capacity to conjugate 4-HNE is attributable to DmGSTS1-1. The high abundance of DmGSTS1-1 (approximately 2% of the soluble protein in adult flies) and its previously reported localization in tissues that are either highly aerobic (indirect flight muscle) or especially sensitive to oxidative damage (neuronal tissue) suggest that the enzyme may have a protective role against deleterious effects of oxidative stress. Such function in insects would be analogous to that carried out in mammals by specialized alpha class glutathione S-transferases (e.g. GSTA4-4). The independent emergence of 4-HNE-conjugating activity in more than one branch of the glutathione S-transferase superfamily suggests that 4-HNE catabolism may be essential for aerobic life.     

Hyodeoxycholate-6-O-glucuronide cannot be quantitated with 3 alpha-hydroxysteroid dehydrogenase

The reaction of the 6-hydroxylated bile acid, hyodeoxycholic acid, and its 6-O-glucuronide conjugate with 3 alpha-hydroxysteroid dehydrogenase was examined. A standard end-point assay and determination of the initial rates of reaction showed only minimal activity of the enzyme toward hyodeoxycholate-6-glucuronide in spite of the presence of a free 3 alpha-hydroxyl group. It was established that 6-hydroxylation itself did not significantly affect the enzyme reaction. It is concluded that the 6-glucuronide either blocks or hinders enzyme access to the 3-hydroxyl group.